The type I interferons (IFNs) regulate the innate immune response to viral infection and have anti-proliferative and pro-apoptotic action which has been exploited in the treatment of cancer. Furthermore, type I IFN signaling is a model for the large family of helical cytokines, which regulate many aspects of proliferation, differentiation and the immune response. Thus, studying type I IFN signaling is expected to impact our understanding of cancer and other diseases and facilitate the development of therapeutic modalities. Type I IFNs can signal through the JAK-STAT pathway. In brief, receptors (IFNaRI and IFNaR2) rely on JAK tyrosine kinases (Tyk2 and Jak1) to initiate signaling. IFN binding triggers JAK activation and phosphorylation of two STATs (Statl and Stat2). The STATs then heterodimerize, complex with the interferon regulatory factor 9 (Irf9), translocate to the nucleus and bind a response element upstream of IFN-regulated genes. Previously, this grant has supported our investigation of multiple aspects of this canonical signaling cascade. Recently, in the course of investigating the interaction between Stat2 and IFNaR2, we found that type I IFNs induce a two-step proteolysis of the IFNaR2 subunit in a manner that resembles the mechanism employed by Notch and the Alzheimer's precursor protein. Cleavage also occurs spontaneously and in response to various stimuli that induce PKC activation. An initial cleavage, mediated by the metalloprotease TACE, releases most of the ectodomain and a second cleavage by the intramembrane presenilin proteases releases the intracellular domain (ICD) of IFNaR2. Preliminary data indicates the ICD is capable of nuclear translocation and that this fragment of IFNaR2 can modulate gene transcription and inhibit cell proliferation, suggesting that type I IFNs might signal via a regulated intramembranous proteolysis (RIP) mechanism. Thus, the overall goal of this renewal application is to determine if type I IFNs can signal, in a physiologically relevant context, via RIP. Moreover, does RIP act in lieu of, or in addition to, the canonical JAK-STAT signaling pathway? Four specific aims are proposed to test this hypothesis. Aim 1 determines if type I IFNs induce cleavage and nuclear translocation of the endogenous IFNaR2 ICD and characterizes the mechanisms(s) initiating production of the ICD. Next, the key experiments in this proposal will test the hypothesis that cleavage is required for physiological effects of IFN by identifying the protease cleavage sites on IFNaR2 (aim 2) and determining if mutations which prevent cleavage perturb the physiological effects of the type I IFNs (aim 3). Finally, aim 4 addresses the mechanism of ICD mediated gene regulation by testing the hypothesis that the ICD functions in a complex with Stat2 and Irf9 to regulate gene transcription.